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cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer)

采用AlphaScreen® 试剂盒(PerkinElmer)进行cAMP累积试验

CK Cassandra Koole
DW Denise Wootten
PS Patrick Sexton

发布: 2013年07月05日第3卷第13期 DOI: 10.21769/BioProtoc.805 浏览次数: 13564

评审: Cheng Zhang

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The authors used this protocol in:

Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Nov 2012

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Abstract

Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing Gs-coupled receptors.

Materials and Reagents

  1. Sterile 96-well clear flat bottom plates (BD Biosciences, Falcon®, catalog number: 353072 )
  2. Phenol Red Free Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 21063-029 )
  3. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  4. 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 )
  5. Tween-20 (Sigma-Aldrich, catalog number: P2287 )
  6. HEPES (Life Technologies, Gibco®, catalog number: 11344-041 )
  7. 100% Ethanol
  8. Milli-Q H2O
  9. Alphascreen® cAMP Assay Kit (PerkinElmer, catalog number: 6760625 )
  10. White OptiPlate-384 well microplate (PerkinElmer, catalog number: 6007290 )
  11. TopSeal (PerkinElmer, catalog number: 6005250 )
  12. Stimulation buffer (see Recipes)
  13. Lysis buffer (see Recipes)
  14. Acceptor buffer (see Recipes)
  15. Donor buffer (see Recipes)

Equipment

  1. Fusion-α plate reader or Envision plate reader with appropriate alphascreen modules (PerkinElmer)
  2. Humidified Incubator
  3. Multichannel Pipettes
  4. Micropipettes
  5. Benchtop centrifuge
  6. Oven
  7. Orbital shake

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Koole, C., Wootten, D. and Sexton, P. M. (2013). cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer) . Bio-protocol 3(13): e805. DOI: 10.21769/BioProtoc.805.

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分类

细胞生物学 > 细胞信号传导 > 第二信使

生物化学 > 蛋白质 > 相互作用 > 蛋白质-配体相互作用

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